Cells interact with structurally distinct types of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What are the differences in cell adhesive structures and biological responses between 2D and 3D matrices, as well as between different types of 3D matrices characteristic of different in vivo microenvironments? 2. What signal transduction mechanisms control cell behavior in different 3D microenvironments? We previously published evidence for the importance of the three-dimensionality of the extracellular matrix surrounding fibroblasts in a variety of cell biological functions including signal transduction, migration, and proliferation. We initiated tests of hypotheses concerning the mechanisms of cell migration in 3D settings. Cells are known to be surrounded by biochemically and structurally distinct matrices in vivo, e.g., a matrix rich in fibronectin fibrils during early craniofacial neural crest migration versus a collagen-rich matrix with varying crosslinking in adult connective tissue. We hypothesized that differences in 3D matrix composition can alter cell migration, the actin cytoskeleton, and/or types of cell adhesion structures. These parameters have not yet been directly compared for normal, non-tumor cells migrating in multiple different 3D matrices. We are currently directly comparing a number of parameters of human fibroblast cell behavior in response to 2D substrates versus four different types of 3D matrix: cell-derived 3D matrix, collagen gels, fibrin gels, and Matrigel. Cells in several of the matrices display distinctive morphology of their cell adhesions and a more peripheral actin cytoskeleton rather than the stress fibers characteristic of cells on 2D substrates. Human fibroblasts showed markedly different capacities for rapid cell migration on these matrices in 2D and 3D;for example, cells migrated the most rapidly on 2D substrates coated with Matrigel, yet they were immobile in 3D Matrigel. Such differences in responses to different 3D matrices while retaining cell adhesion structures will be important to consider when attempting tissue engineering approaches Our previous studies implicated the cytoskeletal protein tensin in fibronectin matrix assembly. In collaboration with Katherine Clark and David Critchley, we continued to compare the functions of the three large tensin isoforms. We found that even though these tensins can compensate surprisingly well for each other in assembly of a fibronectin-based matrix and migration, they have distinctive patterns of localization and adhesion dynamics, as well as different roles in remodeling 3D collagen gels. Tensin 2 was unique among the tensins in its enrichment in dynamic focal adhesions at the leading edge of the cell. Depletion of tensin 2 using siRNA selectively inhibited the ability of human fibroblasts to contract 2D collagen gels. This effect was associated with substantially reduced RhoA activity, and Rho activity was essential for effective gel contraction. In fact, attempting to counter-balance the effects of tensin 2 depletion by siRNA knockdown of DLC1, a RhoGAP tumor suppressor that binds to tensin in focal adhesions and inhibits Rho activation, effectively reversed this inhibition. These studies have identified a novel specific role for tensin 2 in negative regulation of DLC1 to permit Rho-mediated actomyosin contractility and remodeling of a 3D collagen matrix. We are also exploring whether classical models of signal transduction in cell migration established using regular 2D cell culture are valid in the structurally complex 3D environment found in tissues. We have been using tissue explants and in vitro models of 3D extracellular matrix consisting of cell-derived matrix and collagen gels to mimic different types of complex tissue structure while still permitting high-resolution live-cell imaging to visualize intracellular signaling. Our goal is to determine the intracellular organization of signaling pathways in migrating cells in order to understand mechanisms and regulation of cell motility in structurally complex 3D environments. We are placing emphasis on searching for unique modes of migration within different extracellular matrix environments with particular interest in roles of Rho family GTPases. Our preliminary results indicate that normal human fibroblasts can switch between two modes of cell migration, one based on classical lamellipodia at the leading edge of cells, and the other based on a lobopodial mechanism of migration distinct from previously described amoeboid migration. We are examining the involvement of GTPases in these modes of migration. We are continuing to develop methods to visualize cell-surface and cytoskeletal molecular complexes and dynamics in 3D matrices. This technically challenging microscopy technology will be important to develop further in order to allow direct comparisons of cellular functions in 2D versus 3D environments. Understanding whether the nature of the cell biological responses to specific matrix proteins differs in 2D and 3D appears to be important, since initial conclusions from 2D in vitro studies can differ when re-examined under 3D or in vivo conditions.